Human coagulation Factor VII variants

ABSTRACT

The present invention relates to novel human coagulation Factor VIIa variants having coagulant activity as well as nucleic acid constructs encoding such variants, vectors and host cells comprising and expressing the nucleic acid, pharmaceutical compositions, uses and methods of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 09/951,121filed on Sep. 13, 2001 and claims priority under 35 U.S.C. 119 of U.S.application Ser. No. 60/236,455 filed on Sep. 29, 2000 and Danishapplication no. PA 2000 01361 filed on Sep. 13, 2000, the contents ofwhich are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel human coagulation Factor VIIavariants having coagulant activity as well as nucleic acid constructsencoding such variants, vectors and host cells comprising and expressingthe nucleic acid, pharmaceutical compositions, uses and methods oftreatment.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components (or factors) that eventually gives raise to afibrin clot. Generally, the blood components, which participate in whathas been referred to as the coagulation “cascade”, are enzymaticallyinactive proteins (proenzymes or zymogens) that are converted toproteolytic enzymes by the action of an activator (which itself is anactivated clotting factor). Coagulation factors that have undergone sucha conversion are generally referred to as “active factors”, and aredesignated by the addition of the letter “a” to the name of thecoagulation factor (e.g. Factor VIIa).

Initiation of the haemostatic process is mediated by the formation of acomplex between tissue factor, exposed as a result of injury to thevessel wall, and Factor VIIa. This complex then converts Factors IX andX to their active forms. Factor Xa converts limited amounts ofprothrombin to thrombin on the tissue factor-bearing cell. Thrombinactivates platelets and Factors V and VIII into Factors Va and VIIIa,both cofactors in the further process leading to the full thrombinburst. This process includes generation of Factor Xa by Factor IXa (incomplex with factor VIIIa) and occurs on the surface of activatedplatelets. Thrombin finally converts fibrinogen to fibrin resulting information of a fibrin clot. In recent years Factor VII and tissue factorhave been found to be the main initiators of blood coagulation.

Factor VII is a trace plasma glycoprotein that circulates in blood as asingle-chain zymogen. The zymogen is catalytically inactive.Single-chain Factor VII may be converted to two-chain Factor VIIa byFactor Xa, Factor XIIa, Factor IXa, Factor VIIa or thrombin in vitro.Factor Xa is believed to be the major physiological activator of FactorVII. Like several other plasma proteins involved in haemostasis, FactorVII is dependent on Vitamin K for its activity, which is required forthe gamma-carboxylation of multiple glutamic acid residues that areclustered close to the amino terminus of the protein. Thesegamma-carboxylated glutamic acids are required for the metal ion-inducedinteraction of Factor VII with phospholipids. The conversion of zymogenFactor VII into the activated two-chain molecule occurs by cleavage ofan internal Arg₁₅₂-Ile₁₅₃ peptide bond. In the presence of tissuefactor, phospholipids and calcium ions, the two-chain Factor VIIarapidly activates Factor X or Factor IX by limited proteolysis.

It is often desirable to stimulate or improve the coagulation cascade ina subject. Factor VIIa has been used to control bleeding disorders thathave several causes such as clotting factor deficiencies (e.g.haemophilia A and B or deficiency of coagulation Factors XI or VII) orclotting factor inhibitors. Factor VIIa has also been used to controlexcessive bleeding occurring in subjects with a normally functioningblood clotting cascade (no clotting factor deficiencies or inhibitorsagainst any of the coagulation factors). Such bleeding may, for example,be caused by a defective platelet function, thrombocytopenia or vonWillebrand's disease. Bleeding is also a major problem in connectionwith surgery and other forms of tissue damage.

European Patent No. 200,421 (ZymoGenetics) relates to the nucleotidesequence encoding human Factor VII and the recombinant expression ofFactor VII in mammalian cells.

Dickinson et al. (Proc. Natl. Acad. Sci. USA (1996) 93, 14379-14384)relates to Factor VII variants wherein Lys157, Val158, Glu296, Met298,Asp334, Ser336 or Lys337 have been individually replaced by Ala.

Iwanaga et al. (Thromb. Haemost. (supplement August 1999), 466, abstract1474) relates to FVIIa variants wherein residues 316-320 are deleted orresidues 311-322 are replaced with the corresponding residues fromtrypsin.

There is, however, still a need for variants of Factor VIIa havingcoagulant activity, variants with high activity that can be administeredat relatively low doses, and variants which do not produce theundesirable side effects such as systemic activation of the coagulationsystem and bleeding, respectively, associated with conventionaltherapies.

DESCRIPTION OF THE INVENTION

The invention provides coagulation Factor VIIa polypeptides withcoagulant activity.

In a first aspect, the invention provides a Factor VII polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a variant thereof,wherein at least the amino acid corresponding to the Lys at position 157of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala157).

In a second aspect, the invention provides a Factor VII polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a variant thereof,wherein at least the amino acid corresponding to the Lys at position 337of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala337).

In a third aspect, the invention provides a Factor VII polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a variant thereof,wherein at least the amino acid corresponding to the Asp at position 334of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala334).

In a further aspect, the invention provides a Factor VII polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a variant thereof,wherein at least the amino acid corresponding to the Ser at position 336of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala336).

In a further aspect, the invention provides a Factor VII polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a variant thereof,wherein at least the amino acid corresponding to the Val at position 158of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala158).

In a further aspect, the invention provides a Factor VII polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a variant thereof,wherein at least the amino acid corresponding to the Glu at position 296of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala296).

In a further aspect, the invention provides a Factor VII polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a variant thereof,wherein at least the amino acid corresponding to the Met at position 298of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala298).

In a further aspect, the invention provides a Factor VII polypeptidewith the sequence of SEQ ID NO:1, wherein one amino acid independentlyselected from the group consisting of the Lys at position 157, the Lysat position 337, the Asp at position 334, the Ser at position 336, theVal at position 158, the Glu at position 296 and the Met at position 298of SEQ ID NO:1 has been replaced by a different amino acid; with theproviso that the variant is not FVII(Ala157), FVII(Ala334),FVII(Ala336), FVII(Ala337), FVII(Ala158), FVII(Ala296) or FVII(Ala298).

In a further aspect, the invention provides a Factor VII polypeptidewith the sequence of SEQ ID NO:1, wherein two amino acids independentlyselected from the group consisting of the Lys at position 157, the Lysat position 337, the Asp at position 334, the Ser at position 336, theVal at position 158, the Glu at position 296 and the Met at position 298of SEQ ID NO:1 have been replaced by different amino acids.

In a further aspect, the invention provides a Factor VII polypeptidewith the sequence of SEQ ID NO:1, wherein three amino acidsindependently selected from the group consisting of the Lys at position157, the Lys at position 337, the Asp at position 334, the Ser atposition 336, the Val at position 158, the Glu at position 296 and theMet at position 298 of SEQ ID NO:1 have been replaced by different aminoacids.

In a further aspect, the invention provides a Factor VII polypeptidewith the sequence of SEQ ID NO:1, wherein four amino acids independentlyselected from the group consisting of the Lys at position 157, the Lysat position 337, the Asp at position 334, the Ser at position 336, theVal at position 158, the Glu at position 296 and the Met at position 298of SEQ ID NO:1 have been replaced by different amino acids.

In a further aspect, the invention provides a Factor VII polypeptidewith the sequence of SEQ ID NO:1, wherein five amino acids independentlyselected from the group consisting of the Lys at position 157, the Lysat position 337, the Asp at position 334, the Ser at position 336, theVal at position 158, the Glu at position 296 and the Met at position 298of SEQ ID NO:1 have been replaced by different amino acids.

In a further aspect, the invention provides a Factor VII polypeptidewith the sequence of SEQ ID NO:1, wherein six amino acids independentlyselected from the group consisting of the Lys at position 157, the Lysat position 337, the Asp at position 334, the Ser at position 336, theVal at position 158, the Glu at position 296 and the Met at position 298of SEQ ID NO:1 have been replaced by different amino acids.

In a further aspect, the invention provides a Factor VII polypeptidewith the sequence of SEQ ID NO:1, wherein the amino acids consisting ofthe Lys at position 157, the Lys at position 337, the Asp at position334, the Ser at position 336, the Val at position 158, the Glu atposition 296 and the Met at position 298 of SEQ ID NO:1 have beenreplaced by different amino acids.

In a further aspect, the invention provides a coagulation Factor VIIvariant, wherein one or more of the Lys residue in position 157 and theLys residue in position 337 and the Asp residue in position 334 and theSer residue in position 336 and the Val residue in position 158 and theGlu residue in position 296 and the Met residue in position 298 of SEQID NO:1 has(have) been replaced by another amino acid residue which canbe encoded by nucleic acid constructs and, optionally, wherein one ormore additional amino acid residue(s) in the remaining positions in theprotease domain has been replaced by another amino acid residue whichcan be encoded by nucleic acid constructs; with the proviso that thevariant is not FVII(Ala157), FVII(Ala334), FVII(Ala336), FVII(Ala337),FVII(Ala158), FVII(Ala296) or FVII(Ala298).

The term “FVII(Ala157)”, as used herein means a coagulation factor VIIvariant with the sequence of SEQ ID NO:1, wherein the Lys at position157 of SEQ ID NO:1 has been replaced by an alanine. This term does forexample not include coagulation factor VII variants, wherein two or moreamino acids at positions of SEQ ID NO:1 have been replaced by differentamino acids.

The term “FVII(Ala337)”, as used herein means a coagulation factor VIIvariant with the sequence of SEQ ID NO:1, wherein the Lys at position337 of SEQ ID NO:1 has been replaced by an alanine. This term does forexample not include coagulation factor VII variants, wherein two or moreamino acids at positions of SEQ ID NO:1 have been replaced by differentamino acids.

The term “FVII(Ala334)”, as used herein means a coagulation factor VIIvariant with the sequence of SEQ ID NO:1, wherein the Asp at position334 of SEQ ID NO:1 has been replaced by an alanine. This term does forexample not include coagulation factor VII variants, wherein two or moreamino acids at positions of SEQ ID NO:1 have been replaced by differentamino acids.

The term “FVII(Ala336)”, as used herein means a coagulation factor VIIvariant with the sequence of SEQ ID NO:1, wherein the Ser at position336 of SEQ ID NO:1 has been replaced by an alanine. This term does forexample not include coagulation factor VII variants, wherein two or moreamino acids at positions of SEQ ID NO:1 have been replaced by differentamino acids.

The term “FVII(Ala158)”, as used herein means a coagulation factor VIIvariant with the sequence of SEQ ID NO:1, wherein the Val at position158 of SEQ ID NO:1 has been replaced by an alanine. This term does forexample not include coagulation factor VII variants, wherein two or moreamino acids at positions of SEQ ID NO:1 have been replaced by differentamino acids.

The term “FVII(Ala296)”, as used herein means a coagulation factor VIIvariant with the sequence of SEQ ID NO:1, wherein the Glu at position296 of SEQ ID NO:1 has been replaced by an alanine. This term does forexample not include coagulation factor VII variants, wherein two or moreamino acids at positions of SEQ ID NO:1 have been replaced by differentamino acids.

The term “FVII(Ala298)”, as used herein means a coagulation factor VIIvariant with the sequence of SEQ ID NO:1, wherein the Met at position298 of SEQ ID NO:1 has been replaced by an alanine. This term does forexample not include coagulation factor VII variants, wherein two or moreamino acids at positions of SEQ ID NO:1 have been replaced by differentamino acids.

In a further aspect, the invention relates to a nucleic acid constructencoding a Factor VII polypeptide of the invention.

In a further aspect, the invention provides a nucleic acid constructcomprising a nucleotide sequence encoding a Factor VII variant.

In a further aspect, the invention provides a recombinant vectorcomprising the nucleic acid construct encoding a FVIIa variant.

In a further aspect, the invention provides a recombinant host cellcomprising the nucleic acid construct or the vector.

In a further aspect, the invention provides a transgenic animalcontaining and expressing the nucleic acid construct.

In a further aspect, the invention provides a transgenic plantcontaining and expressing the nucleic acid construct.

In a further aspect, the invention relates to a method for producing theFactor VII polypeptide of the invention, the method comprisingcultivating a cell comprising the nucleic acid construct in anappropriate growth medium under conditions allowing expression of thenucleic acid construct and recovering the resulting polypeptide from theculture medium.

In a further aspect, the invention provides a method for producing theFactor VII variant of the invention, the method comprising cultivating acell comprising the nucleic acid construct in an appropriate growthmedium under conditions allowing expression of the nucleic acidconstruct and recovering the resulting polypeptide from the culturemedium.

In a further aspect, the invention relates to a method for producing theFactor VII polypeptide, the method comprising recovering the polypeptidefrom milk produced by the transgenic animal.

In a further aspect, the invention provides a method for producing theFactor VII variant, the method comprising recovering the variant frommilk produced by the transgenic animal.

In a further aspect, the invention relates to a method for producing theFactor VII polypeptide, the method comprising cultivating a cell of atransgenic plant comprising the nucleic acid construct, and recoveringthe polypeptide from the resulting plant.

In a further aspect, the invention provides a method for producing theFactor VII variant, the method comprising cultivating a cell of atransgenic plant comprising the nucleic acid construct, and recoveringthe variant from the resulting plant.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a variant thereof, wherein at least theamino acid corresponding to the Lys at position 157 of SEQ ID NO:1 hasbeen replaced by a different amino acid; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a variant thereof, wherein at least theamino acid corresponding to the Lys at position 337 of SEQ ID NO:1 hasbeen replaced by a different amino acid; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a variant thereof, wherein at least theamino acid corresponding to the Asp at position 334 of SEQ ID NO:1 hasbeen replaced by a different amino acid; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a variant thereof, wherein at least theamino acid corresponding to the Ser at position 336 of SEQ ID NO:1 hasbeen replaced by a different amino acid; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a variant thereof, wherein at least theamino acid corresponding to the Val at position 158 of SEQ ID NO:1 hasbeen replaced by a different amino acid; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a variant thereof, wherein at least theamino acid corresponding to the Glu at position 296 of SEQ ID NO:1 hasbeen replaced by a different amino acid; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a Factor VII polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a variant thereof, wherein at least theamino acid corresponding to the Met at position 298 of SEQ ID NO:1 hasbeen replaced by a different amino acid; and, optionally, apharmaceutically acceptable carrier.

In a further aspect, the invention provides a pharmaceutical compositioncomprising a coagulation Factor VII variant, wherein one or more of theLys residue in position 157 and the Lys residue in position 337 and theAsp residue in position 334 and the Ser residue in position 336 and theVal residue in position 158 and the Glu residue in position 296 and theMet residue in position 298 of SEQ ID NO:1 has(have) been replaced byanother amino acid residue which can be encoded by nucleic acidconstructs and, optionally, wherein one or more additional amino acidresidue(s) in the remaining positions in the protease domain has beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs; and, optionally, a pharmaceutically acceptable carrier.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theLys at position 157 of SEQ ID NO:1 has been replaced by a differentamino acid; for the preparation of a medicament for the treatment ofbleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theLys at position 337 of SEQ ID NO:1 has been replaced by a differentamino acid; for the preparation of a medicament for the treatment ofbleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theAsp at position 334 of SEQ ID NO:1 has been replaced by a differentamino acid; for the preparation of a medicament for the treatment ofbleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theSer at position 336 of SEQ ID NO:1 has been replaced by a differentamino acid; for the preparation of a medicament for the treatment ofbleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theVal at position 158 of SEQ ID NO:1 has been replaced by a differentamino acid; for the preparation of a medicament for the treatment ofbleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theGlu at position 296 of SEQ ID NO:1 has been replaced by a differentamino acid; for the preparation of a medicament for the treatment ofbleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, the invention relates to the use of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theMet at position 298 of SEQ ID NO:1 has been replaced by a differentamino acid; for the preparation of a medicament for the treatment ofbleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, the invention provides the use of a coagulationFactor VII variant, wherein one or more of the Lys residue in position157 and the Lys residue in position 337 and the Asp residue in position334 and the Ser residue in position 336 and the Val residue in position158 and the Glu residue in position 296 and the Met residue in position298 of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs and, optionally, whereinone or more additional amino acid residue(s) in the remaining positionsin the protease domain has been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs; for the preparation ofa medicament for the treatment of bleeding episodes or for theenhancement of the normal haemostatic system.

In a further aspect, the invention relates to a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theLys at position 157 of SEQ ID NO:1 has been replaced by a differentamino acid; to a subject in need thereof.

In a further aspect, the invention relates to a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theLys at position 337 of SEQ ID NO:1 has been replaced by a differentamino acid; to a subject in need thereof.

In a further aspect, the invention relates to a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theAsp at position 334 of SEQ ID NO:1 has been replaced by a differentamino acid; to a subject in need thereof.

In a further aspect, the invention relates to a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theSer at position 336 of SEQ ID NO:1 has been replaced by a differentamino acid; to a subject in need thereof.

In a further aspect, the invention relates to a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theVal at position 158 of SEQ ID NO:1 has been replaced by a differentamino acid; to a subject in need thereof.

In a further aspect, the invention relates to a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theGlu at position 296 of SEQ ID NO:1 has been replaced by a differentamino acid; to a subject in need thereof.

In a further aspect, the invention relates to a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a Factor VIIpolypeptide comprising the amino acid sequence of SEQ ID NO:1 or avariant thereof, wherein at least the amino acid corresponding to theMet at position 298 of SEQ ID NO:1 has been replaced by a differentamino acid; to a subject in need thereof.

In a further aspect, the invention provides a method for the treatmentof bleeding episodes in a subject or for the enhancement of the normalhaemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a coagulationFactor VII variant, wherein one or more of the Lys residue in position157 and the Lys residue in position 337 and the Asp residue in position334 and the Ser residue in position 336 and the Val residue in position158 and the Glu residue in position 296 and the Met residue in position298 of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs and, optionally, whereinone or more additional amino acid residue(s) in the remaining positionsin the protease domain has been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs; to a subject in needthereof.

In a further aspect, the invention provides a pharmaceutical compositioncomprising a coagulation Factor VII variant, wherein one or more of theLys residue in position 157 and the Lys residue in position 337 and theAsp residue in position 334 and the Ser residue in position 336 and theVal residue in position 158 and the Glu residue in position 296 and theMet residue in position 298 of SEQ ID NO:1 has(have) been replaced byanother amino acid residue which can be encoded by nucleic acidconstructs and, optionally, wherein one or more additional amino acidresidue(s) in the remaining positions in the protease domain has beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs; with the proviso that the variant is not FVII(Ala157),FVII(Ala334), FVII(Ala336), FVII(Ala337), FVII(Ala158), FVII(Ala296) orFVII(Ala298); and optionally, a pharmaceutically acceptable carrier.

In a further aspect, the invention provides the use of a coagulationFactor VII variant, wherein one or more of the Lys residue in position157 and the Lys residue in position 337 and the Asp residue in position334 and the Ser residue in position 336 and the Val residue in position158 and the Glu residue in position 296 and the Met residue in position298 of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs and, optionally, whereinone or more additional amino acid residue(s) in the remaining positionsin the protease domain has been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs; with the proviso thatthe variant is not FVII(Ala157), FVII(Ala334), FVII(Ala336),FVII(Ala337), FVII(Ala158), FVII(Ala296) or FVII(Ala298); for thepreparation of a medicament for the treatment of bleeding episodes orfor the enhancement of the normal haemostatic system.

In a further aspect, the invention provides a method for the treatmentor prophylaxis of bleeding disorders in a subject or for the enhancementof the normal haemostatic system, the method comprising administering atherapeutically or prophylactically effective amount of a coagulationFactor VII variant, wherein one or more of the Lys residue in position157 and the Lys residue in position 337 and the Asp residue in position334 and the Ser residue in position 336 and the Val residue in position158 and the Glu residue in position 296 and the Met residue in position298 of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs and, optionally, whereinone or more additional amino acid residue(s) in the remaining positionsin the protease domain has been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs; with the proviso thatthe variant is not FVII(Ala157), FVII(Ala334), FVII(Ala336),FVII(Ala337), FVII(Ala158), FVII(Ala296) or FVII(Ala298); to a subjectin need thereof.

In one embodiment of the invention, the factor VII polypeptide is apolypeptide, wherein the amino acid corresponding to the Lys at position157 of SEQ ID NO:1 has been replaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid corresponding to the Lys atposition 337 of SEQ ID NO:1 has been replaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid corresponding to the Asp atposition 334 of SEQ ID NO:1 has been replaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid corresponding to the Ser atposition 336 of SEQ ID NO:1 has been replaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid corresponding to the Val atposition 158 of SEQ ID NO:1 has been replaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid corresponding to the Glu atposition 296 of SEQ ID NO:1 has been replaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid corresponding to the Met atposition 298 of SEQ ID NO:1 has been replaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid in the remainingpositions in the protease domain has been replaced by a different aminoacid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at the most 20 additional amino acids in theremaining positions in the protease domain have been replaced bydifferent amino acids.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid corresponding to an aminoacid at a position selected from 159-170 of SEQ ID NO:1 has beenreplaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid corresponding to an aminoacid at a position selected from 290-312 of SEQ ID NO:1 has beenreplaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein at least one amino acid corresponding to an aminoacid at a position selected from 330-339 of SEQ ID NO:1 has beenreplaced by a different amino acid.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Lys at position 157 of SEQ ID NO:1 has beenreplaced by an amino acid selected from the group consisting of Gly,Val, Ser, Thr, Asn, Gln, Asp, and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Lys at position 337 of SEQ ID NO:1 has beenreplaced by an amino acid selected from the group consisting of Ala,Gly, Val, Ser, Thr, Asn, Gln, Asp, and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Asp at position 334 of SEQ ID NO:1 has beenreplaced by an amino acid selected from the group consisting of Gly, andGlu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Ser at position 336 of SEQ ID NO:1 has beenreplaced by an amino acid selected from the group consisting of Gly, andGlu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Val at position 158 of SEQ ID NO:1 has beenreplaced by an amino acid selected from the group consisting of Ser,Thr, Asn, Gln, Asp, and Glu.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Glu at position 296 of SEQ ID NO:1 has beenreplaced by an amino acid selected from the group consisting of Arg,Lys, and Val.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Met at position 298 of SEQ ID NO:1 has beenreplaced by an amino acid selected from the group consisting of Lys,Arg, Gln, and Asn.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein the amino acid has been replaced by a differentamino acid which can be encoded by nucleic acid constructs.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Factor VII polypeptide is human Factor VII.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide, wherein said Factor VII polypeptide is human Factor VIIa.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide independently selected from the group consisting of[E296V]-FVII, [M298Q]-FVII, and [S336G]-FVII.

In a further embodiment of the invention, the factor VII polypeptide isa polypeptide independently selected from the group consisting of[V158T/M298Q]-FVII, [E296V/M298Q]-FVII, [V158D/E296V]-FVII,[V158D/M298Q]-FVII, and [V 158D/M298K]-FVII.

In a further embodiment of the invention, the factor VII polypeptide is[V158D/E296V/M298Q]-FVII.

In a further embodiment of the invention, the factor VII polypeptide is[V158D/E296V/M298Q/K337A]-FVII.

In a further embodiment, the factor VII variants are variants whereinone or more of the Lys residue in position 157 and the Lys residue inposition 337 and the Asp residue in position 334 and the Ser residue inposition 336 and the Val residue in position 158 and the Glu residue inposition 296 and the Met residue in position 298 of SEQ ID NO:1has(have) been replaced by another amino acid residue which can beencoded by nucleic acid constructs and, optionally, wherein one or moreadditional amino acid residue(s) in the remaining positions in theprotease domain has been replaced by another amino acid residue whichcan be encoded by nucleic acid constructs;

with the proviso that the variant is not FVII(Ala157), FVII(Ala334),FVII(Ala336), FVII(Ala337), FVII(Ala158), FVII(Ala296) or FVII(Ala298).

In one embodiment, the factor VII variants are variants wherein one ormore of the Lys residue in position 157 and the Lys residue in position337 and the Asp residue in position 334 and the Ser residue in position336 and the Val residue in position 158 and the Glu residue in position296 and the Met residue in position 298 of SEQ ID NO:1 has(have) beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs; with the proviso that the variant is not FVII(Ala157),FVII(Ala334), FVII(Ala336), FVII(Ala337), FVII(Ala158), FVII(Ala296) orFVII(Ala298).

In a further embodiment, one or more of the Lys residue in position 157and the Lys residue in position 337 of SEQ ID NO:1 has(have) beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs.

In a further embodiment, one or more of the Lys residue in position 157and the Val residue in position 158 and the Glu residue in position 296and the Met residue in position 298 of SEQ ID NO:1 has(have) beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs.

In a further embodiment, the Lys residue in position 157 and one or moreof the Val residue in position 158 and the Glu residue in position 296and the Met residue in position 298 of SEQ ID NO:1 has(have) beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs.

In a further embodiment, one or more of the Lys residue in position 157and the Asp residue in position 334 and the Ser residue in position 336of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs.

In a further embodiment, the Lys residue in position 157 and one or moreof the Asp residue in position 334 and the Ser residue in position 336of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs.

In a further embodiment, one or more of the Lys residue in position 337and the Val residue in position 158 and the Glu residue in position 296and the Met residue in position 298 of SEQ ID NO:1 has(have) beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs.

In a further embodiment, the Lys residue in position 337 and one or moreof the Val residue in position 158 and the Glu residue in position 296and the Met residue in position 298 of SEQ ID NO:1 has(have) beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs.

In a further embodiment, one or more of the Lys residue in position 337and the Asp residue in position 334 and the Ser residue in position 336of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs.

In a further embodiment, the Lys residue in position 337 and one or moreof the Asp residue in position 334 and the Ser residue in position 336of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs.

In a further embodiment, one or more of the Val residue in position 158and the Glu residue in position 296 and the Met residue in position 298and one or more of the Asp residue in position 334 and the Ser residuein position 336 of SEQ ID NO:1 has(have) been replaced by another aminoacid residue which can be encoded by nucleic acid constructs.

In a further embodiment, the Lys residue in position 157 of SEQ ID NO:1has(have) been replaced by another amino acid residue which can beencoded by nucleic acid constructs.

In a further embodiment, the Lys residue in position 337 of SEQ ID NO:1has(have) been replaced by another amino acid residue which can beencoded by nucleic acid constructs.

In a further embodiment, one or more of the Val residue in position 158and the Glu residue in position 296 and the Met residue in position 298of SEQ ID NO:1 has(have) been replaced by another amino acid residuewhich can be encoded by nucleic acid constructs.

In a further embodiment, one or more of the Asp residue in position 334and the Ser residue in position 336 of SEQ ID NO:1 has(have) beenreplaced by another amino acid residue which can be encoded by nucleicacid constructs.

In a further embodiment, the Lys residue in position 157 has beenreplaced by Gly, Val, Ser, Thr, Asn, Gln, Asp or Glu; and/or the Lysresidue in position 337 has been replaced by Gly, Val, Ser, Thr, Asn,Gln, Asp or Glu; and/or the Val residue in position 158 has beenreplaced by Ser, Thr, Asn, Gln, Asp or Glu; and/or the Glu residue inposition 296 has been replaced by Arg, Lys or Val; and/or the Metresidue in position 298 has been replaced by Arg, Lys, Gln or Asn;and/or the Asp residue in position 334 has been replaced by Glu; and/orthe Ser residue in position 336 has been replaced by Gly.

In a further embodiment the Lys residue in position 157 or the Lysresidue in position 337 or the Asp residue in position 334 or the Serresidue in position 336 is the only amino acid residue that has beenreplaced.

In a further embodiment, the Val residue in position 158 and the Gluresidue in position 296 and the Met residue in position 298 are the onlyamino acid residues that have been replaced.

In a further embodiment, the ratio between the activity of a variantaccording to the invention and the activity of the native Factor VIIpolypeptide shown in SEQ ID NO:1 is at least about 1.25 when tested inthe “In Vitro Hydrolysis Assay” defined herein. In another embodiment,the ratio is at least about 2.0; in another embodiment, at least about4.0.

In a further embodiment, the Gln residue in position 312 in the proteasedomain has not been replaced.

In a further embodiment, the recombinant host cell is of mammalianorigin. In another embodiment, the cell is selected from the groupconsisting of CHO cells, BHK cells or HEK cells.

In a further embodiment, at the most 20 additional amino acid residuesin the remaining positions in the protease domain (positions 153-406)have been replaced. In one embodiment, at the most 15 additional aminoacid residues are replaced; in another embodiment, at the most 10 aminoacid residues are replaced; in another embodiment, at the most 5 aminoacid residues are replaced.

In a further embodiment, one or more of the amino acid residues inpositions 157, 337, 158, 296, 298, 334, or 336 is/are the only aminoacid residue(s) that has/have been replaced.

In a further embodiment, one or both of the Lys residues in position 157and in position 337 has/have been replaced by a neutral amino acidresidue, or one of the residues has been replaced by a negativelycharged amino acid residue, or one Lys residue has been replaced by aneutral amino acid residue and one Lys residue has been replaced by anegatively charged amino acid residue.

In a further embodiment, one or both of the Asp residue in position 334and the Ser residue in position 336 has/have been replaced by an aminoacid residue that is able to form hydrogen bonds and/or able to form asalt bridge, or one or both of the residues is/are replaced by a smallamino acid residue.

In a further embodiment, the Lys residue in position 157 and/or the Lysresidue in position 337 is/are the only amino acid residue(s) thathas/have been replaced. In one embodiment, the Lys residue in position157 has been replaced. In another embodiment, the Lys residue inposition 337 has been replaced.

In a further embodiment, the Val residue in position 158 and/or the Metresidue in position 298 is/are the only amino acid residue(s) thathas/have been replaced.

In a further embodiment, the Asp residue in position 334 and/or the Serresidue in position 336 is/are the only amino acid residue(s) thathas/have been replaced.

In a further embodiment, the Lys residue at position 157 has beenreplaced by an amino acid residue selected from a list of Val, Leu, Ile,Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Arg, His, Asp,and Gln. In another embodiment, the Lys residue in position 157 has beenreplaced by an amino acid residue selected from the group consisting ofGly, Val, Ser, Thr, Asn, Gln, Asp, and Glu.

In a further embodiment, Lys residue at position 337 has been replacedby an amino acid residue selected from a list of Val, Leu, Ile, Met,Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Arg, His, Asp or Gln.In another embodiment, the Lys residue in position 337 has been replacedby Gly, Val, Ser, Thr, Asn, Gln, Asp or Glu.

In a further embodiment, Val residue in position 158 has been replacedby an amino acid residue selected from a list of Leu, Ile, Met, Phe,Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.In a further embodiment, the Val residue in position 158 has beenreplaced by an amino acid residue selected from the group consisting ofSer, Thr, Asn, Gln, Asp and Glu.

In a further embodiment, the Glu residue in position 296 has beenreplaced by an amino acid residue selected from a list of Val, Leu, Ile,Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Lys, Arg, His, Asp orGln. In a further embodiment, the residue has been replaced by Arg, Lysor Val.

In a further embodiment, the Met residue in position 298 has beenreplaced by an amino acid residue selected from a list of Val, Leu, Ile,Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Lys, Arg, His, Glu, Asp orGln. In another embodiment, the residue has been replaced by Lys, Arg,Gln or Asn.

In a further embodiment, the Asp residue in position 334 has beenreplaced by Gly and Glu.

In a further embodiment, the Ser residue in position 336 that has beenreplaced by Gly and Glu.

In a further embodiment of the invention the ratio between the activityof the variant and the activity of the native Factor VII polypeptideshown in SEQ ID NO:1 is at least about 1.25 when tested in the “In VitroHydrolysis Assay” as defined herein (Example 11). In another embodiment,the ratio is at least about 2.0; in yet another embodiment, at leastabout 4.0.

In a further aspect, the invention provides FVIIa variants that haveincreased tissue factor-independent activity compared to native FVIIa.In one embodiment thereof, the increased activity is not accompanied bychanges in the substrate specificity. In one embodiment, the binding ofthe variants to tissue factor are not impaired (compared to wild-typeFVIIa); in another embodiment, the variants have at least the activityof wild-type Factor VIIa when bound to tissue factor.

In a further embodiment, the Factor VII variants, in addition to thealready performed amino acid replacement in positions 157, 158, 296,298, 334, 336 or 337 and the optional amino acid replacements elsewherein the protease domain, also have some amino acid residues replaced inthe N-terminal Gla domain (residues 1-37). In one embodiment, one ormore of the amino acid residues in positions 10 and 32 (referring to SEQID NO:1) of Factor VII is/are replaced with another amino acid residuethat can be encoded by nucleic acid constructs. In one embodiment, theamino acid residue Pro in position 10 is replaced by Gln, Arg, His, Gln,Asn or Lys; and/or the amino acid residue Lys in position 32 is replacedby Glu, Gln or Asn.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the full amino acid sequence of native human coagulationFactor VII (SEQ ID NO:1).

In the present specification, amino acids are represented usingabbreviations, as indicated in table 1, approved by IUPAC-IUB Commissionon Biochemical Nomenclature (CBN). Amino acid and the like havingisomers represented by name or the following abbreviations are innatural L-form unless otherwise indicated. Further, the left and rightends of an amino acid sequence of a peptide are, respectively, the N-and C-termini unless otherwise specified. TABLE 1 Abbreviations foramino acids: Amino acid Tree-letter code One-letter code Glycine Gly GProline Pro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile IMethionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr YTryptophan Trp W Histidine His H Lysine Lys K Arginine Arg R GlutamineGln Q Asparagine Asn N Glutamic Glu E Acid Aspartic Acid Asp D SerineSer S Threonine Thr T

It has now been found that FVIIa variants wherein at least one of theamino acid residues Lys157, Val158, Glu296, Met298, Asp334, Ser336 orLys337 (and optionally one or more additional residues) is/are replacedby another amino acid residue have coagulant activity.

The residues are located in an area believed to affect the insertion ofthe amino terminus of the protease domain and thereby the formation ofthe catalytically active conformation of Factor VIIa which is dependenton a salt bridge between the terminal amino group of Ile 153 and theside chain of Asp343. The replacements may remove electrostaticrepulsions, add hydrogen bonds or otherwise facilitate the insertion ofthe amino terminus.

Due to the higher inherent activity of the described Factor VIIa variantcompared to native FVIIa, a lower dose is foreseen to be adequate toobtain a functionally adequate concentration at the site of action andthus it will be possible to administer a lower dose to the subjecthaving bleeding episodes or needing enhancement of the normalhaemostatic system.

As discussed briefly above, it is hypothesized by the present inventorsthat by replacing one or more of the Lys residue in position 157 and theLys residue in position 337 and the Val residue in position 158 and theGlu residue in position 296 and the Met residue in position 298 and theAsp residue in position 334 and the Ser residue in position 336, FactorVIIa will spontaneously attain a more active conformation that normallyhas to be induced by tissue factor. Such Factor VIIa variants exhibit aninherent activity which may be therapeutically useful in situationswhere the procoagulant activity is independent of tissue factor (FactorXa generation on the platelet surface) such as for example, when highdoses of NovoSeven® are administered.

Replacement of additional amino acid residues in the protease domainmay, in addition to the effect obtained by replacement of one or more ofthe Lys residue in position 157 and the Lys residue in position 337 andthe Val residue in position 158 and the Glu residue in position 296 andthe Met residue in position 298 and the Asp residue in position 334 andthe Ser residue in position 336, further facilitate formation of theactive conformation of the molecule. It is believed, however, that themost pronounced effects will be seen when the above-mentioned mutationsare carried out in the vicinity (sequential or three-dimensional) ofthese seven residues.

Replacement of a few amino acid residues in the N-terminal Gla domain(residues 1-37) of Factor VII may provide the protein with asubstantially higher affinity for membrane phospholipids, such asmembrane phospholipids of tissue factor-bearing cells or of platelets.Thus, the Factor VII variants mentioned above may, in addition to thealready performed amino acid replacement in positions 157, 158, 296,298, 334, 336 or 337 and the optional amino acid replacements elsewherein the protease domain, also have some amino acid residues replaced inthe N-terminal Gla domain, thereby obtaining a protein having anincreased activity as well as an increased affinity for membranephospholipids compared to native Factor VII. Preferably the amino acidresidues in positions 10 and 32 (referring to SEQ ID NO:1) of Factor VIImay be replaced with another amino acid residue that can be encoded bynucleic acid constructs. Examples of preferred amino acid residues to beincorporated in the above-mentioned positions are: The amino acidresidue Pro in position 10 is replaced by Gln, Arg, His, Gln, Asn orLys; and/or the amino acid residue Lys in position 32 is replaced byGlu, Gln or Asn.

Other residues in the Gla domain, based on the different phospholipidaffinities and sequences of the vitamin K-dependent plasma proteins, mayalso be considered for substitution. In the present context thethree-letter indications of the amino acids have been used in theirconventional meaning. Unless indicated explicitly, the amino acidsmentioned herein are L-amino acids.

The term “N-terminal GLA-domain” means the amino acid sequence 1-37 ofFVII.

The term “protease domain” means the amino acid sequence 153-406 of FVII(the heavy chain of FVIIa).

The three-letter indication “GLA” means 4-carboxyglutamic acid(γ-carboxyglutamate).

The term “neutral amino acid residue” (at pH 6-8) is intended tocomprise amino acids selected from the list of Ala, Val, Leu, Ile, Pro,Met, Phe, Trp, Gly, Ser, Thr, Cys, Tyr, Asn, Gln.

The term “small amino acid” is intended to comprise amino acids selectedfrom Gly, Glu and Ala.

The term “negatively charged amino acid residue” (at pH 6-8) is intendedto comprise amino acids selected from Asp and Glu.

The term “Factor VII polypeptide” as used herein means any proteincomprising the amino acid sequence 1-406 of native human Factor VII (SEQID NO:1) or variants thereof. This includes but are not limited to humanFactor VII, human Factor VIIa and variants thereof.

The term “Factor VII” as used herein is intended to comprise theinactive one-chain zymogen Factor VII molecule as well as the activatedtwo-chain Factor VII molecule (Factor VIIa). This includes proteins thathave the amino acid sequence 1-406 of native human Factor VII or FactorVIIa. It also includes proteins with a slightly modified amino acidsequence, for instance, a modified N-terminal end including N-terminalamino acid deletions or additions so long as those proteinssubstantially retain the activity of Factor VIIa. The term “factorVIIa”, or “FVIIa” as used herein means a product consisting of theactivated form (factor VIIa). “Factor VII” or “Factor VIIa” within theabove definition also includes natural allelic variations that may existand occur from one individual to another. Also, degree and location ofglycosylation or other post-translation modifications may vary dependingon the chosen host cells and the nature of the host cellularenvironment.

The terms “variant” or “variants”, as used herein, is intended todesignate Factor VII having the sequence of SEQ ID NO:1, wherein one ormore amino acids of the parent protein have been substituted by anotheramino acid and/or wherein one or more amino acids of the parent proteinhave been deleted and/or wherein one or more amino acids have beeninserted in protein and/or wherein one or more amino acids have beenadded to the parent protein. Such addition can take place either at theN-terminal end or at the C-terminal end of the parent protein or both.The “variant” or “variants” within this definition have FVII activity inits activated two-chain molecular form. In one embodiment a variant is65% identical with the sequence of of SEQ ID NO:1. In one embodiment avariant is 80% identical with the sequence of of SEQ ID NO:1. In anotherembodiment a variant is 90% identical with the sequence of of SEQ IDNO:1. In a further embodiment a variant is 95% identical with thesequence of of SEQ ID NO:1.

As used herein the term “nucleic acid construct” is intended to mean anynucleic acid molecule of cDNA, genomic DNA, synthetic DNA, semisynthetic DNA, RNA origin or mixed origin. The term “construct” isintended to indicate a nucleic acid segment which may be single- ordouble-stranded, and which may be based on a complete or partialnaturally occurring nucleotide sequence encoding the polypeptide ofinterest. The construct may optionally contain other nucleic acidsegments. In a similar way, the term “amino acid residue which can beencoded by nucleic acid constructs” covers amino acid residues which canbe encoded by the nucleic acid constructs defined above, i.e. aminoacids such as Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr,Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

The term “a different amino acid” as used herein means an amino acidthat are different from the amino acid naturally present at thatposition. This includes but are not limited to amino acids that can beencoded by a nucleic acid construct. Preferably the different amino acidis in natural L-form and can be encoded by a nucleic acid construct. Aspecific example being L-cysteine (Cys).

In the present context, the term “treatment” is meant to include bothprevention of an expected bleeding, such as in surgery, and regulationof an already occurring bleeding, such as in trauma, with the purpose ofinhibiting or minimising the bleeding. Prophylactic administration ofthe Factor VIIa variant according to the invention is thus included inthe term “treatment”.

The term “activity” as used herein means the ability of a Factor VIIpolypeptide or a variant thereof to convert its substrate Factor X tothe active Factor Xa. The activity of a Factor VII polypeptide may bemeasured with the “In Vitro Proteolysis Assay”.

The term “inherent activity” also includes the ability to generatethrombin on the surface of activated platelets in the absence of tissuefactor.

The term “enhancement of the normal haemostatic system” means anenhancement of the ability to generate thrombin.

As used herein the term “bleeding disorder” reflects any defect,congenital, acquired or induced, of cellular or molecular origin that ismanifested in bleedings. Examples are clotting factor deficiencies (e.g.haemophilia A and B or deficiency of coagulation Factors XI or VII),clotting factor inhibitors, defective platelet function,thrombocytopenia or von Willebrand's disease.

The term “bleeding episodes” is meant to include uncontrolled andexcessive bleeding which is a major problem both in connection withsurgery and other forms of tissue damage. Uncontrolled and excessivebleeding may occur in subjects having a normal coagulation system andsubjects having coagulation or bleeding disorders. Clotting factordeficiencies (haemophilia A and B, deficiency of coagulation factors XIor VII) or clotting factor inhibitors may be the cause of bleedingdisorders. Excessive bleedings also occur in subjects with a normallyfunctioning blood clotting cascade (no clotting factor deficiencies or-inhibitors against any of the coagulation factors) and may be caused bya defective platelet function, thrombocytopenia or von Willebrand'sdisease. In such cases, the bleedings may be likened to those bleedingscaused by haemophilia because the haemostatic system, as in haemophilia,lacks or has abnormal essential clotting “compounds” (such as plateletsor von Willebrand factor protein) that causes major bleedings. Insubjects who experience extensive tissue damage in association withsurgery or vast trauma, the normal haemostatic mechanism may beoverwhelmed by the demand of immediate haemostasis and they may developbleeding in spite of a normal haemostatic mechanism. Achievingsatisfactory haemostasis also is a problem when bleedings occur inorgans such as the brain, inner ear region and eyes with limitedpossibility for surgical haemostasis. The same problem may arise in theprocess of taking biopsies from various organs (liver, lung, tumourtissue, gastrointestinal tract) as well as in laparoscopic surgery.Common for all these situations is the difficulty to provide haemostasisby surgical techniques (sutures, clips, etc.) which also is the casewhen bleeding is diffuse (haemorrhagic gastritis and profuse uterinebleeding). Acute and profuse bleedings may also occur in subjects onanticoagulant therapy in whom a defective haemostasis has been inducedby the therapy given. Such subjects may need surgical interventions incase the anticoagulant effect has to be counteracted rapidly. Radicalretropubic prostatectomy is a commonly performed procedure for subjectswith localized prostate cancer. The operation is frequently complicatedby sigrnificant and sometimes massive blood loss. The considerable bloodloss during prostatectomy is mainly related to the complicatedanatomical situation, with various densely vascularized sites that arenot easily accessible for surgical haemostasis, and which may result indiffuse bleeding from a large area. Another situation that may causeproblems in the case of unsatisfactory haemostasis is when subjects witha normal haemostatic mechanism are given anticoagulant therapy toprevent thromboembolic disease. Such therapy may include heparin, otherforms of proteoglycans, warfarin or other forms of vitamin K-antagonistsas well as aspirin and other platelet aggregation inhibitors.

In one embodiment of the invention, the bleeding is associated withhaemophilia. In another embodiment, the bleeding is associated withhaemophilia with aquired inhibitors. In another embodiment, the bleedingis associated with thrombocytopenia. In another embodiment, the bleedingis associated with von Willebrand's disease. In another embodiment, thebleeding is associated with severe tissue damage. In another embodiment,the bleeding is associated with severe trauma. In another embodiment,the bleeding is associated with surgery. In another embodiment, thebleeding is associated with laparoscopic surgery. In another embodiment,the bleeding is associated with haemorrhagic gastritis. In anotherembodiment, the bleeding is profuse uterine bleeding. In anotherembodiment, the bleeding is occurring in organs with a limitedpossibility for mechanical haemostasis. In another embodiment, thebleeding is occurring in the brain, inner ear region or eyes. In anotherembodiment, the bleeding is associated with the process of takingbiopsies. In another embodiment, the bleeding is associated withanticoagulant therapy.

The term “subject” as used herein is intended to mean any animal, inparticular mammals, such as humans, and may, where appropriate, be usedinterchangeably with the term “patient”.

As used herein the term “appropriate growth medium” means a mediumcontaining nutrients and other components required for the growth ofcells and the expression of the nucleic acid sequence encoding theFactor VII variant of the invention.

Preparation of Factor VII Variants

The Factor VII variants described herein may be produced by means ofrecombinant nucleic acid techniques. In general, a cloned wild-typeFactor VII nucleic acid sequence is modified to encode the desiredprotein. This modified sequence is then inserted into an expressionvector, which is in turn transformed or transfected into host cells.Higher eukaryotic cells, in particular cultured mammalian cells, arepreferred as host cells. The complete nucleotide and amino acidsequences for human Factor VII are known (see U.S. Pat. No. 4,784,950,where the cloning and expression of recombinant human Factor VII isdescribed). The bovine Factor VII sequence is described in Takeya etal., J. Biol. Chem. 263:14868-14872 (1988)).

The amino acid sequence alterations may be accomplished by a variety oftechniques. Modification of the nucleic acid sequence may be bysite-specific mutagenesis. Techniques for site-specific mutagenesis arewell known in the art and are described in, for example, Zoller andSmith (DNA 3:479-488, 1984) or “Splicing by extension overlap”, Hortonet al., Gene 77, 1989, pp. 61-68. Thus, using the nucleotide and aminoacid sequences of Factor VII, one may introduce the alteration(s) ofchoice. Likewise, procedures for preparing a DNA construct usingpolymerase chain reaction using specific primers are well known topersons skilled in the art (cf. PCR Protocols, 1990, Academic Press, SanDiego, Calif., USA).

The nucleic acid construct encoding the Factor VII variant of theinvention may suitably be of genomic or cDNA origin, for instanceobtained by preparing a genomic or cDNA library and screening for DNAsequences coding for all or part of the polypeptide by hybridizationusing synthetic oligonucleotide probes in accordance with standardtechniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

The nucleic acid construct encoding the Factor VII variant may also beprepared synthetically by established standard methods, e.g. thephosphoamidite method described by Beaucage and Caruthers, TetrahedronLetters 22 (1981), 1859-1869, or the method described by Matthes et al.,EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method,oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser,purified, annealed, ligated and cloned in suitable vectors.

Furthermore, the nucleic acid construct may be of mixed synthetic andgenomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

The nucleic acid construct is preferably a DNA construct,

DNA sequences for use in producing Factor VII variants according to thepresent invention will typically encode a pre-pro polypeptide at theamino-terminus of Factor VII to obtain proper posttranslationalprocessing (e.g. gamma-carboxylation of glutamic acid residues) andsecretion from the host cell. The pre-pro polypeptide may be that ofFactor VII or another vitamin K-dependent plasma protein, such as FactorIX, Factor X, prothrombin, protein C or protein S. As will beappreciated by those skilled in the art, additional modifications can bemade in the amino acid sequence of the Factor VII variants where thosemodifications do not significantly impair the ability of the protein toact as a coagulant. For example, the Factor VII variants can also bemodified in the activation cleavage site to inhibit the conversion ofzymogen Factor VII into its activated two-chain form, as generallydescribed in U.S. Pat. No. 5,288,629.

Expression vectors for use in expressing Factor VIIa variants willcomprise a promoter capable of directing the transcription of a clonedgene or cDNA. Preferred promoters for use in cultured mammalian cellsinclude viral promoters and cellular promoters. Viral promoters includethe SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981)and the CMV promoter (Boshart et al., Cell 41:521-530, 1985). Aparticularly preferred viral promoter is the major late promoter fromadenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982).Cellular promoters include the mouse kappa gene promoter (Bergman etal., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983) and the mouse V_(H)promoter (Loh et al., Cell 33:85-93, 1983). A particularly preferredcellular promoter is the mouse metallothionein-1 promoter (Palmiter etal., Science 222:809-814, 1983). Expression vectors may also contain aset of RNA splice sites located downstream from the promoter andupstream from the insertion site for the Factor VII sequence itself.Preferred RNA splice sites may be obtained from adenovirus and/orimmunoglobulin genes. Also contained in the expression vectors is apolyadenylation signal located downstream of the insertion site.Particularly preferred polyadenylation signals include the early or latepolyadenylation signal from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the adenovirus 5 Elb region, the humangrowth hormone gene terminator (DeNoto et al. Nucl. Acids Res.9:3719-3730, 1981) or the polyadenylation signal from the human FactorVII gene or the bovine Factor VII gene. The expression vectors may alsoinclude a noncoding viral leader sequence, such as the adenovirus 2tripartite leader, located between the promoter and the RNA splicesites; and enhancer sequences, such as the SV40 enhancer.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass., incorporated herein by reference). The person skilledin the art will easily be able to choose suitable selectable markers.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If, on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as “carrier DNA,” to the mixture that isintroduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically for 1-2 days, to begin expressing the gene ofinterest. The medium used to culture the cells may be any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. in catalogues of the American Type Culture Collection).The media are prepared using procedures known in the art (see, e.g.,references for bacteria and yeast; Bennett, J. W. and LaSure, L.,editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).Growth media generally include a carbon source, a nitrogen source,essential amino acids, essential sugars, vitamins, salts, phospholipids,proteins and growth factors. For production of gamma-carboxylated FactorVII variants, the medium will contain vitamin K, preferably at aconcentration of about 0.1 mg/ml to about 5 mg/ml. Drug selection isthen applied to select for the growth of cells that are expressing theselectable marker in a stable fashion. For cells that have beentransfected with an amplifiable selectable marker the drug concentrationmay be increased to select for an increased copy number of the clonedsequences, thereby increasing expression levels. Clones of stablytransfected cells are then screened for expression of the desired FactorVII variant.

Preferred mammalian cell lines include the CHO (ATCC CCL 61), COS-1(ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL 1573;Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferredBHK cell line is the tk⁻ ts13 BHK cell line (Waechter and Baserga, Proc.Natl. Acad. Sci. USA 79:1106-1110, 1982), hereinafter referred to as BHK570 cells. The BHK 570 cell line is available from the American TypeCulture Collection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCCaccession number CRL 10314. A tk⁻ ts13 BHK cell line is also availablefrom the ATCC under accession number CRL 1632. In addition, a number ofother cell lines may be used, including Rat Hep I (Rat hepatoma; ATCCCRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).

Transgenic animal technology may be employed to produce the Factor VIIvariants of the invention. It is preferred to produce the proteinswithin the mammary glands of a host female mammal. Expression in themammary gland and subsequent secretion of the protein of interest intothe milk overcomes many difficulties encountered in isolating proteinsfrom other sources. Milk is readily collected, available in largequantities, and biochemically well characterized. Furthermore, the majormilk proteins are present in milk at high concentrations (typically fromabout 1 to 15 g/l).

From a commercial point of view, it is clearly preferable to use as thehost a species that has a large milk yield. While smaller animals suchas mice and rats can be used (and are preferred at the proof ofprinciple stage), it is preferred to use livestock mammals including,but not limited to, pigs, goats, sheep and cattle. Sheep areparticularly preferred due to such factors as the previous history oftransgenesis in this species, milk yield, cost and the readyavailability of equipment for collecting sheep milk (see, for example,WO 88/00239 for a comparison of factors influencing the choice of hostspecies). It is generally desirable to select a breed of host animalthat has been bred for dairy use, such as East Friesland sheep, or tointroduce dairy stock by breeding of the transgenic line at a laterdate. In any event, animals of known, good health status should be used.

To obtain expression in the mammary gland, a transcription promoter froma milk protein gene is used. Milk protein genes include those genesencoding caseins (see U.S. Pat. No. 5,304,489), beta-lactoglobulin,a-lactalbumin, and whey acidic protein. The beta-lactoglobulin (BLG)promoter is preferred. In the case of the ovine beta-lactoglobulin gene,a region of at least the proximal 406 bp of 5′ flanking sequence of thegene will generally be used, although larger portions of the 5′ flankingsequence, up to about 5 kbp, are preferred, such as a ˜4.25 kbp DNAsegment encompassing the 5′ flanking promoter and non-coding portion ofthe beta-lactoglobulin gene (see Whitelaw et al., Biochem. J. 286: 31-39(1992)). Similar fragments of promoter DNA from other species are alsosuitable.

Other regions of the beta-lactoglobulin gene may also be incorporated inconstructs, as may genomic regions of the gene to be expressed. It isgenerally accepted in the art that constructs lacking introns, forexample, express poorly in comparison with those that contain such DNAsequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840(1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991);Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO91/02318, each of which is incorporated herein by reference). In thisregard, it is generally preferred, where possible, to use genomicsequences containing all or some of the native introns of a geneencoding the protein or polypeptide of interest, thus the furtherinclusion of at least some introns from, e.g, the beta-lactoglobulingene, is preferred. One such region is a DNA segment that provides forintron splicing and RNA polyadenylation from the 3′ non-coding region ofthe ovine beta-lactoglobulin gene. When substituted for the natural 3′non-coding sequences of a gene, this ovine beta-lactoglobulin segmentcan both enhance and stabilize expression levels of the protein orpolypeptide of interest. Within other embodiments, the regionsurrounding the initiation ATG of the variant Factor VII sequence isreplaced with corresponding sequences from a milk specific protein gene.Such replacement provides a putative tissue-specific initiationenvironment to enhance expression. It is convenient to replace theentire variant Factor VII pre-pro and 5′ non-coding sequences with thoseof, for example, the BLG gene, although smaller regions may be replaced.

For expression of Factor VII variants in transgenic animals, a DNAsegment encoding variant Factor VII is operably linked to additional DNAsegments required for its expression to produce expression units. Suchadditional segments include the above-mentioned promoter, as well assequences that provide for termination of transcription andpolyadenylation of mRNA. The expression units will further include a DNAsegment encoding a secretory signal sequence operably linked to thesegment encoding modified Factor VII. The secretory signal sequence maybe a native Factor VII secretory signal sequence or may be that ofanother protein, such as a milk protein (see, for example, von Heijne,Nucl. Acids Res. 14: 4683-4690 (1986); and Meade et al., U.S. Pat. No.4,873,316, which are incorporated herein by reference).

Construction of expression units for use in transgenic animals isconveniently carried out by inserting a variant Factor VII sequence intoa plasmid or phage vector containing the additional DNA segments,although the expression unit may be constructed by essentially anysequence of ligations. It is particularly convenient to provide a vectorcontaining a DNA segment encoding a milk protein and to replace thecoding sequence for the milk protein with that of a variant Factor VIIpolypeptide; thereby creating a gene fusion that includes the expressioncontrol sequences of the milk protein gene. In any event, cloning of theexpression units in plasmids or other vectors facilitates theamplification of the variant Factor VII sequence. Amplification isconveniently carried out in bacterial (e.g. E. coli) host cells, thusthe vectors will typically include an origin of replication and aselectable marker functional in bacterial host cells. The expressionunit is then introduced into fertilized eggs (including early-stageembryos) of the chosen host species. Introduction of heterologous DNAcan be accomplished by one of several routes, including microinjection(e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science240: 1468-1474 (1988)) or site-directed integration using embryonic stem(ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539(1992)). The eggs are then implanted into the oviducts or uteri ofpseudopregnant females and allowed to develop to term. Offspringcarrying the introduced DNA in their germ line can pass the DNA on totheir progeny in the normal, Mendelian fashion, allowing the developmentof transgenic herds. General procedures for producing transgenic animalsare known in the art (see, for example, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986;Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol.Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8: 140-143(1990); Ebert et al., Bio/Technology 9: 835-838 (1991); Krimpenfort etal., Bio/Technology 9: 844-847 (1991); Wall et al., J. Cell. Biochem.49: 113-120 (1992); U.S. Pat. No. 4,873,191; U.S. Pat. No. 4,873,316; WO88/00239, WO 90/05188, WO 92/11757; and GB 87/00458). Techniques forintroducing foreign DNA sequences into mammals and their germ cells wereoriginally developed in the mouse (see, e.g., Gordon et al., Proc. Natl.Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214:1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985);Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); andHogan et al. (ibid.)). These techniques were subsequently adapted foruse with larger animals, including livestock species (see, e.g., WO88/00239, WO 90/05188, and WO 92/11757; and Simons et al.,Bio/Technology 6: 179-183 (1988)). To summarise, in the most efficientroute used to date in the generation of transgenic mice or livestock,several hundred linear molecules of the DNA of interest are injectedinto one of the pro-nuclei of a fertilized egg according to establishedtechniques. Injection of DNA into the cytoplasm of a zygote can also beemployed.

Production in transgenic plants may also be employed. Expression may begeneralised or directed to a particular organ, such as a tuber (see,Hiatt, Nature 344:469-479 (1990); Edelbaum et al., J. Interferon Res.12:449-453 (1992); Sijmons et al., Bio/Technology 8:217-221 (1990); andEP 0 255 378).

The Factor VII variants of the invention are recovered from cell culturemedium or milk. The Factor VII variants of the present invention may bepurified by a variety of procedures known in the art including, but notlimited to, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989). Preferably, they may be purified by affinity chromatographyon an anti-Factor VII antibody column. The use of calcium-dependentmonoclonal antibodies, as described by Wakabayashi et al., J. Biol.Chem. 261:11097-11108, (1986) and Thim et al., Biochemistry 27:7785-7793, (1988), is particularly preferred. Additional purificationmay be achieved by conventional chemical purification means, such ashigh performance liquid chromatography. Other methods of purification,including barium citrate precipitation, are known in the art, and may beapplied to the purification of the novel Factor VII variants describedherein (see, for example, Scopes, R., Protein Purification,Springer-Verlag, N.Y., 1982).

For therapeutic purposes it is preferred that the Factor VII variants ofthe invention are substantially pure. Thus, in a preferred embodiment ofthe invention the Factor VII variants of the invention is purified to atleast about 90 to 95% homogeneity, preferably to at least about 98%homogeneity. Purity may be assessed by e.g. gel electrophoresis andamino-terminal amino acid sequencing.

The Factor VII variant is cleaved at its activation site in order toconvert it to its two-chain form. Activation may be carried outaccording to procedures known in the art, such as those disclosed byOsterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No.4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983); orKisiel and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983). Alternatively,as described by Bjoern et al. (Research Disclosure, 269 September 1986,pp. 564-565), Factor VII may be activated by passing it through anion-exchange chromatography column, such as Mono Q® (Pharmacia fineChemicals) or the like. The resulting activated Factor VII variant maythen be formulated and administered as described below.

Assays

The invention also provides suitable assays for selecting preferredFactor VIIa variants according to the invention. These assays can beperformed as a simple preliminary in vitro test.

Thus, Example 11 herein discloses a simple test (entitled “In VitroHydrolysis Assay”) for the activity of Factor VIIa variants of theinvention. Based thereon, Factor VIIa variants which are of particularinterest are such variants where the ratio between the activity of thevariant and the activity of native Factor VII shown in FIG. 1 is above1.0, e.g. at least about 1.25, preferably at least about 2.0, such as atleast about 3.0 or, even more preferred, at least about 4.0 when testedin the “In Vitro Hydrolysis Assay” defined herein.

The activity of the variants can also be measured using a physiologicalsubstrate such as factor X (In Vitro Proteolysis Assay, see Example 12),suitably at a concentration of 100-1000 nM, where the factor Xagenerated is measured after the addition of a suitable chromogenicsubstrate (eg. S-2765). In addition, the activity assay may be run atphysiological temperature.

The ability of the FVIIa variants to generate thrombin can also bemeasured in an assay comprising all relevant coagulation factors andinhibitors at physiological concentrations (minus factor VIII whenmimicking hemophilia A conditions) and activated platelets (as describedon p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 whichis hereby incorporated as reference).

Administration and Pharmaceutical Compositions

The Factor VII variants according to the present invention may be usedto control bleeding disorders which have several causes such as clottingfactor deficiencies (e.g. haemophilia A and B or deficiency ofcoagulation factors XI or VII) or clotting factor inhibitors, or theymay be used to control excessive bleeding occurring in subjects with anormally functioning blood clotting cascade (no clotting factordeficiencies or inhibitors against any of the coagulation factors). Thebleedings may be caused by a defective platelet function,thrombocytopenia or von Willebrand's disease. They may also be seen insubjects in whom an increased fibrinolytic activity has been induced byvarious stimuli.

In subjects who experience extensive tissue damage in association withsurgery or vast trauma, the haemostatic mechanism may be overwhelmed bythe demand of immediate haemostasis and they may develop bleedings inspite of a normal haemostatic mechanism. Achieving satisfactoryhaemostasis is also a problem when bleedings occur in organs such as thebrain, inner ear region and eyes and may also be a problem in cases ofdiffuse bleedings (haemorrhagic gastritis and profuse uterine bleeding)when it is difficult to identify the source. The same problem may arisein the process of taking biopsies from various organs (liver, lung,tumour tissue, gastrointestinal tract) as well as in laparoscopicsurgery. These situations share the difficulty of providing haemostasisby surgical techniques (sutures, clips, etc.). Acute and profusebleedings may also occur in subjects on anticoagulant therapy in whom adefective haemostasis has been induced by the therapy given. Suchsubjects may need surgical interventions in case the anticoagulanteffect has to be counteracted rapidly. Another situation that may causeproblems in the case of unsatisfactory haemostasis is when subjects witha normal haemostatic mechanism are given anticoagulant therapy toprevent thromboembolic disease. Such therapy may include heparin, otherforms of proteoglycans, warfarin or other forms of vitamin K-antagonistsas well as aspirin and other platelet aggregation inhibitors.

A systemic activation of the coagulation cascade may lead todisseminated intravascular coagulation (DIC). However, suchcomplications have not been seen in subjects treated with high doses ofrecombinant FVIIa because of a localised haemostatic process of the kindinduced by the complex formation between FVIIa and TF exposed at thesite of vessel wall injury. The Factor VII variants according to theinvention may thus also be used in their activated form to control suchexcessive bleedings associated with a normal haemostatic mechanism.

For treatment in connection with deliberate interventions, the FactorVII variants of the invention will typically be administered withinabout 24 hours prior to performing the intervention, and for as much as7 days or more thereafter. Administration as a coagulant can be by avariety of routes as described herein.

The dose of the Factor VII variants ranges from about 0.05 mg to 500mg/day, preferably from about 1 mg to 200 mg/day, and more preferablyfrom about 10 mg to about 175 mg/day for a 70 kg subject as loading andmaintenance doses, depending on the weight of the subject and theseverity of the condition.

The pharmaceutical compositions are primarily intended for parenteraladministration for prophylactic and/or therapeutic treatment.Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly,or it may be administered by continuous or pulsatile infusion. Thecompositions for parenteral administration comprise the Factor VIIvariant of the invention in combination with, preferably dissolved in, apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, such as water, buffered water,0.4% saline, 0.3% glycine and the like. The Factor VII variants of theinvention can also be formulated into liposome preparations for deliveryor targeting to the sites of injury. Liposome preparations are generallydescribed in, e.g., U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,501,728,and U.S. Pat. No. 4,975,282. The compositions may be sterilised byconventional, well-known sterilisation techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilised, the lyophilised preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc.

The concentration of Factor VII variant in these formulations can varywidely, i.e., from less than about 0.5% by weight, usually at or atleast about 1% by weight to as much as 15 or 20% by weight and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution and 10mg of the Factor VII variant. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa.(1990).

The compositions containing the Factor VII variants of the presentinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic applications, compositions are administeredto a subject already suffering from a disease, as described above, in anamount sufficient to cure, alleviate or partially arrest the disease andits complications. An amount adequate to accomplish this is defined as“therapeutically effective amount”. As will be understood by the personskilled in the art amounts effective for this purpose will depend on theseverity of the disease or injury as well as the weight and generalstate of the subject. In general, however, the effective amount willrange from about 0.05 mg up to about 500 mg of the Factor VII variantper day for a 70 kg subject, with dosages of from about 1.0 mg to about200 mg of the Factor VII variant per day being more commonly used.

It must be kept in mind that the materials of the present invention maygenerally be employed in serious disease or injury states, that is, lifethreatening or potentially life threatening situations. In such cases,in view of the minimisation of extraneous substances and general lack ofimmunogenicity of human Factor VII variants in humans, it is possibleand may be felt desirable by the treating physician to administer asubstantial excess of these variant Factor VII compositions.

In prophylactic applications, compositions containing the Factor VIIvariant of the invention are administered to a subject susceptible to orotherwise at risk of a disease state or injury to enhance the subject'sown coagulative capability. Such an amount is defined to be a“prophylactically effective dose.” In prophylactic applications, theprecise amounts once again depend on the subject's state of health andweight, but the dose generally ranges from about 0.05 mg to about 500 mgper day for a 70-kilogram subject, more commonly from about 1.0 mg toabout 200 mg per day for a 70-kilogram subject.

Single or multiple administrations of the compositions can be carriedout with dose levels and patterns being selected by the treatingphysician. For ambulatory subjects requiring daily maintenance levels,the Factor VII variants may be administered by continuous infusion usinge.g. a portable pump system.

Local delivery of the Factor VII variant of the present invention, suchas, for example, topical application may be carried out, for example, bymeans of a spray, perfusion, double balloon catheters, stent,incorporated into vascular grafts or stents, hydrogels used to coatballoon catheters, or other well established methods. In any event, thepharmaceutical compositions should provide a quantity of Factor VIIvariant sufficient to effectively treat the subject.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES

The terminology for amino acid substitutions used the following examplesare as follows. The first letter represent the amino acid naturallypresent at a position of SEQ ID NO:1. The following number represent theposition in SEQ ID NO:1. The second letter represent the different aminoacid substituting for (replacing) the natural amino acid. An example isM298Q, where an methionine at position 298 of SEQ ID NO:1 is replaced bya glutamine. In another example, V158T/M298Q, the valine in position 158of SEQ ID NO:1 is replaced by a threonine and the methionine in position298 of SEQ ID NO:1 is replaced by a Glutamine in the same Factor VIIpolypeptide.

Example 1

DNA Encoding [V158T/M298Q]-FVII, [K157A]-FVII, [E296V]-FVII,[E296V/M298Q]-FVII and [V158D/E296V]-FVII, [V158D/M298Q]-FVII,[V158D/M298K]-FVII, [V158D/E296V/M298Q]-FVII, [M298Q]-FVII,[S336G]-FVII, [K337A]-FVII, [V158D/E296V/M2980/K337A]-FVII.

A DNA construct encoding [V158T/M298Q]-FVII, [K157A]-FVII, [E296V]-FVII,[E296V/M298Q]-FVII and [V158D/E296V]-FVII, [V158D/M298Q]-FVII,[V158D/M298K]-FVII, [V158D/E296V/M298Q]-FVII, [M298Q]-FVII,[V158D/E296V/M298Q/K337A]-FVII, [S336G]-FVII, and [K337A]-FVII wereprepared by site-directed mutagenesis using a supercoiled, doublestranded DNA vector with an insert of interest and two synthetic primerscontaining the desired mutation. The following primer pairs were used:For [K157A]-FVII: 5′-CCG AAT TGT GGG GGG CGC GGT GTG CCC CAA AGG G-3′(SEQ ID NO:2) 5′-CCC TTT GGG GCA CAC CGC GCC CCC CAC AAT TCG G-3′ (SEQID NO:3) For [V158D]-FVII: 5′-GTG GGG GGC AAG GAG TGC CCC AAA GGG G-3′(SEQ ID NO:4) 5′-CCC CTT TGG GGC AGT CCT TGC CCC CCA C-3′ (SEQ ID NO:5)For [V158T]-FVII: 5′-GTG GGG GGC AAG ACG TGC CCC AAA GGG G-3′ (SEQ IDNO:6) 5′-CCC CTT TGG GGC ACG TCT TGC CCC CCA C-3′ (SEQ ID NO:7) For[E296V/M298Q]-FVII: 5′-GCC ACG GCC CTG GTG CTC CAG GTC CTC AAC GTGCCC-3′ (SEQ ID NO:8) 5′-GGG CAC GTT GAG GAC CTG GAG CAC CAG GGC CGTGGC-3′ (SEQ ID NO:9) For [M298Q]-FVII: 5′-GCC CTG GAG CTC CAG GTC CTCAAC GTG CCC-3′ (SEQ ID NO:10) 5′-GGG CAC GTT GAG GAC CTG GAG CTC CAGGGC-3′ (SEQ ID NO:11) For [M298K]-FVII: 5′-GCC CTG GAG CTC AAG GTC CTCAAC GTG-3′ (SEQ ID NO:12) 5′-CAC CTT GAG GAC CTT GAG CTC CAG GGC-3′ (SEQID NO:13) For [S336G]-FVII: 5′-GGC TAC TCG GAT GGC GGC AAG GAC TCC TGCAAG-3′ (SEQ ID NO:14) 5′-CTT GCA GGA GTC CTT GCC GCC ATC CGA GTA GCC-3′(SEQ ID NO:15) For [K337A]-FVII: 5′-CGG ATG GCA GCG CGG ACT CCT GCA AGGG-3′ (SEQ ID NO:16) 5′-CCC TTG CAG GAG TCC GCG CTG CCA TCC G-3′ (SEQ IDNO:17)

The oligonucleotide primers, each complementary to opposite strands ofthe vector, were extended during temperature cycling by means of Pfu DNApolymerase. On incorporation of the primers, a mutated plasmidcontaining staggered nicks was generated. Following temperature cycling,the product was treated with DpnI which is specific for methylated andhemimethylated DNA to digest the parental DNA template and to select formutation-containing synthesized DNA.

Procedures for preparing a DNA construct using polymerase chain reactionusing specific primers are well known to persons skilled in the art (cf.PCR Protocols, 1990, Academic Press, San Diego, Calif., USA).

Example 2

Preparation of [V158T/M298Q]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant[V158T/M298Q]-FVII. The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-mi column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [V158T/M298Q]-FVII were pooled and appliedto a 25-ml column containing monoclonal antibody F1A2 (Novo Nordisk,Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B (PharmaciaBiotech). The column was equilibrated with 50 mM Hepes, pH 7.5,containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [V158T/M298Q]-FVII was transferred to aCa²⁺-containing buffer. The yield of each step was followed by factorVII ELISA measurements and the purified protein was analysed bySDS-PAGE.

Example 3

Preparation of [K157A]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant [K157A]-FVII.The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [K157A]-FVII were pooled and applied to a25-ml column containing monoclonal antibody F1A2 (Novo Nordisk,Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B (PharmaciaBiotech). The column was equilibrated with 50 mM Hepes, pH 7.5,containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [K157A]-FVII was transferred to a Ca²⁺-containingbuffer. The yield of each step was followed by factor VII ELISAmeasurements and the purified protein was analysed by SDS-PAGE.

Example 4

Preparation of [V158D/M298Q]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant[V158D/M298Q]-FVII. The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [V158D/M298Q]-FVII were pooled and appliedto a 25-ml column containing monoclonal antibody F1A2 (Novo Nordisk,Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B (PharmaciaBiotech). The column was equilibrated with 50 mM Hepes, pH 7.5,containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [V158D/M298Q]-FVII was transferred to aCa²⁺-containing buffer. The yield of each step was followed by factorVII ELISA measurements and the purified protein was analysed bySDS-PAGE.

Example 5

Preparation of [V158D/M298K]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant[V158D/M298K]-FVII. The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [V158D/M298K]-FVII were pooled and appliedto a 25-ml column containing monoclonal antibody F1A2 (Novo Nordisk,Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B (PharmaciaBiotech). The column was equilibrated with 50 mM Hepes, pH 7.5,containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [V158D/M298K]-FVII was transferred to aCa²⁺-containing buffer. The yield of each step was followed by factorVII ELISA measurements and the purified protein was analysed bySDS-PAGE.

Example 6

Preparation of [V158D/E296V/M298Q]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant[V158D/E296V/M298Q]-FVII. The Factor VII variant was purified asfollows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [V158D/E296V/M298Q]-FVII were pooled andapplied to a 25-ml column containing monoclonal antibody F1A2 (NovoNordisk, Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B(Pharmacia Biotech). The column was equilibrated with 50 mM Hepes, pH7.5, containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [V158D/E296V/M298Q]-FVII was transferred to aCa²⁺-containing buffer. The yield of each step was followed by factorVII ELISA measurements and the purified protein was analysed bySDS-PAGE.

Example 7

Preparation of [M298Q]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant [M298Q]-FVII.The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [M298Q]-FVII were pooled and applied to a25-ml column containing monoclonal antibody F1A2 (Novo Nordisk,Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B (PharmaciaBiotech). The column was equilibrated with 50 mM Hepes, pH 7.5,containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [M298Q]-FVII was transferred to a Ca²⁺-containingbuffer. The yield of each step was followed by factor VII ELISAmeasurements and the purified protein was analysed by SDS-PAGE.

Example 8

Preparation of [S336G]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant [S336G]-FVII.The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [S336G]-FVII were pooled and applied to a25-ml column containing monoclonal antibody F1A2 (Novo Nordisk,Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B (PharmaciaBiotech). The column was equilibrated with 50 mM Hepes, pH 7.5,containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [S336G]-FVII was transferred to a Ca²⁺-containingbuffer. The yield of each step was followed by factor VII ELISAmeasurements and the purified protein was analysed by SDS-PAGE.

Example 9

Preparation of [K337A]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385,241-243) to obtain expression of the variant [K337A]-FVII. TheFactor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [K337A]-FVII were pooled and applied to a25-ml column containing monoclonal antibody F1A2 (Novo Nordisk,Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B (PharmaciaBiotech). The column was equilibrated with 50 mM Hepes, pH 7.5,containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [K337A]-FVII was transferred to a Ca²⁺-containingbuffer. The yield of each step was followed by factor VII ELISAmeasurements and the purified protein was analysed by SDS-PAGE.

Example 10

Preparation of [V158D/E296V/M298Q/K337A]-FVII.

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant[V158D/E296V/M298Q/K337A]-FVII. The Factor VII variant was purified asfollows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by stepping from 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100,pH 8.0 to 10 mM Tris, 50 mM NaCl, 25 mM CaCl₂, 0.1% Triton X-100, pH8.0. The fractions containing [V158D/E296V/M298Q/K337A]-FVII were pooledand applied to a 25-ml column containing monoclonal antibody F1A2 (NovoNordisk, Bagsvaerd, Denmark) coupled to CNBr-activated Sepharose 4B(Pharmacia Biotech). The column was equilibrated with 50 mM Hepes, pH7.5, containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [V158D/E296V/M298Q/K337A]-FVII was transferred to aCa²⁺-containing buffer. The yield of each step was followed by factorVII ELISA measurements and the purified protein was analysed bySDS-PAGE.

Example 11

In Vitro Hydrolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) are assayed in parallel to directlycompare their specific activities. The assay is carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrateD-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), finalconcentration 1 mM, is added to Factor VIIa (final concentration 100 nM)in 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/mlbovine serum albumin. The absorbance at 405 nm is measured continuouslyin a SpectraMax™ 340 plate reader (Molecular Devices, USA). Theabsorbance developed during a 20-minute incubation, after subtraction ofthe absorbance in a blank well containing no enzyme, is used tocalculate the ratio between the activities of variant and wild-typeFactor VIIa:Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Example 12

In Vitro Proteolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) are assayed in parallel to directlycompare their specific activities. The assay is carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin, are incubated for15 min. Factor X cleavage is then stopped by the addition of 50 microL50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/mlbovine serum albumin. The amount of Factor Xa generated is measured byaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during 10minutes, after subtraction of the absorbance in a blank well containingno FVIIa, is used to calculate the ratio between the proteolyticactivities of variant and wild-type Factor VIIa:Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Example 13

Relative Activities of FVIIa Variants Measured in the Assays Describedin Examples 11 and 12 Ratio in Ratio in Variant example 11 example 12K157A 0.9 Not determined V158T/M298Q-FVIIa 3.8 10 V158D/M298Q-FVIIa 2.02.7 V158D/M298K-FVIIa 0.3 Not determined V158D/E296V/M298Q-FVIIa 7.8 28M298Q-FVIIa 3.4 5.5 V158D/E296V/M298Q/K337A-FVIIa 11.0 47 S336G-FVIIa0.6 Not determined K337A-FVIIa 3.9 4.4 wt-FVIIa 1.0 1.0

1. A nucleic acid construct comprising a nucleotide sequence encoding aFactor VII variant polypeptide comprising a substitution relative to thesequence of SEQ ID No:1 selected from the group consisting of: (a)substitution of Val158 with any amino acid other than Ala; (b)substitution of Glu296 with any amino acid other than Ala; and (c)combinations of the foregoing.
 2. A nucleic acid construct as defined inclaim 1, wherein said construct is a vector.
 3. A recombinant host cellcomprising a nucleic acid construct as defined in claim
 1. 4. A cell asdefined in claim 3, wherein said cell is selected from the groupconsisting of CHO, BHK, and HEK cells.
 5. A method for producing avariant VII polypeptide, said method comprising (i) cultivating a cellas defined in claim 3 in an appropriate growth medium under conditionsallowing expression of the nucleic acid construct and (ii) recoveringthe resulting polypeptide from the culture medium.
 6. A nucleic acidconstruct as defined in claim 1, wherein said variant further comprisesat least one substitution selected from the group consisting of: (d)substitution of Lys157 with any amino acid other than Ala; (e)substitution of Lys337 with any amino acid other than Ala; (f)substitution of Asp334 with any amino acid other than Ala; (g)substitution of Ser336 with any amino acid other than Ala; and (h)combinations of any of the foregoing.
 7. A nucleic acid construct asdefined in claim 1, wherein the variant is selected from the groupconsisting of V158T+M298Q-FVII, E296V+M298Q-FVII, V158D+E296V-FVII,V158D+M298Q-FVII, and V158D+M298K-FVII.
 8. A nucleic acid construct asdefined in claim 1, wherein the variant is V158D+E296V+M298Q-FVII.
 9. Amethod for producing a variant VII polypeptide, said method comprising(i) cultivating a cell comprising a nucleic acid construct as defined inclaim 6 in an appropriate growth medium under conditions allowingexpression of the nucleic acid and (ii) recovering the resultingpolypeptide from the culture medium.
 10. A method for producing avariant VII polypeptide, said method comprising (i) cultivating a cellcomprising a nucleic acid construct as defined in claim 7 in anappropriate growth medium under conditions allowing expression of thenucleic acid and (ii) recovering the resulting polypeptide from theculture medium.
 11. A method for producing a variant VII polypeptide,said method comprising (i) cultivating a cell comprising a nucleic acidconstruct as defined in claim 8 in an appropriate growth medium underconditions allowing expression of the nucleic acid and (ii) recoveringthe resulting polypeptide from the culture medium.